G Protein Activation by Human Dopamine D3 Receptors in High-Expressing Chinese Hamster Ovary Cells: A Guanosine-59-O-(3-[S]thio)-Triphosphate Binding and Antibody Study

Despite extensive study, the G protein coupling of dopamine D3 receptors is poorly understood. In this study, we used guanosine59-O-(3-[S]thio)-triphosphate ([S]-GTPgS) binding to investigate the activation of G proteins coupled to human (h) D3 receptors stably expressed in Chinese hamster ovary (CHO) cells. Although the receptor expression level was high (15 pmol/mg), dopamine only stimulated G protein activation by 1.6-fold. This was despite the presence of marked receptor reserve for dopamine, as revealed by Furchgott analysis after irreversible hD3 receptor inactivation with the alkylating agent, EEDQ (Nethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline). Thus, halfmaximal stimulation of [S]-GTPgS binding required only 11.8% receptor occupation of hD3 sites. In contrast, although the hD2(short) receptor expression level in another CHO cell line was 11-fold lower, stimulation by dopamine was higher (2.5-fold). G protein activation was increased at hD3 and, less potently, at hD2 receptors by the preferential D3 agonists, PD 128,907 [(1)(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] and (1)-7-OH-DPAT (7-hydroxy-2(di-n-propylamino)tetralin). Furthermore, the selective D3 antagonists, S 14297 ((1)-[7-(N, N-dipropylamino)-5,6,7,8tetrahydro-naphtho(2,3b)dihydro-2,3-furane]) and GR 218,231 (2(R,S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)1,2,3,4tetrahydronaphtalene), blocked dopamine-stimulated [S]GTPgS binding more potently at hD3 than at hD2 sites. Antibodies against Gai/ao reduced dopamine-induced G protein activation at both CHO-hD3 and -hD2 membranes, whereas GaS antibodies had no effect at either site. In contrast, incubation with anti-Gaq/a11 antibodies, which did not affect dopamine-induced G protein activation at hD2 receptors, attenuated hD3-induced G protein activation. These data suggest that hD3 receptors may couple to Gaq/a11 and would be consistent with the observation that pertussis toxin pretreatment, which inactivates only Gi/o proteins, only submaximally (80%) blocked dopamine-stimulated [S]GTPgS binding in CHO-hD3 cells. Taken together, the present data indicate that 1) hD3 receptors functionally couple to G protein activation in CHO cells, 2) hD3 receptors activate G proteins less effectively than hD2 receptors, and 3) hD3 receptors may couple to different G protein subtypes than hD2 receptors, including nonpertussis sensitive Gq/11 proteins. Dopaminergic neurotransmission is mediated by five receptor subtypes (D1 to D5) which can be grouped into two receptor families. D1-like receptors include the D1 and D5 subtypes, whereas D2-like receptors include the D2, D3, and D4 subtypes. D2 and D3 receptors, in particular, display marked sequence homology and pharmacological similarity in their in vitro ligand binding profiles (Levant, 1997; Missale et al., 1998). However, D3 receptors may be distinguished from D2 receptors by several factors. D3 receptors are concentrated in limbic rather than striatal brain regions (Liu et al., 1996; Hall et al., 1996). Furthermore, they mediate stimulation, rather than inhibition, of c-fos expression in striatal neurones (Pilon et al., 1994; Morris et al., 1997), and inhibition, rather than stimulation, of locomotor activity in rats (Svensson et al., 1994; Starr and Starr, 1995). In addition, whereas D2 receptors couple efficiently to second-messenger systems, markedly inhibiting adenylyl cyclase activity, such responses have proved elusive and complex for D3 receptors (e.g., Freedman et al., 1994; MacKenzie et al., 1994; Tang et al., 1994; Griffon et al., 1997). Indeed, D3 receptors couple selectively to inhibition of adenylyl cyclase type V, but not type I or VI, and only weakly to type II (Robinson and ABBREVIATIONS: EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; GR 218,231, 2(R,S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4tetrahydronaphtalene; [S]GTPgS, guanosine-59-O-(3-[S]thio)-triphosphate; (1)7-OH-DPAT, 7-hydroxy-2-(di-n-propylamino)tetralin; PD 128,907, (1)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol; S 14297, (1)-[7-(N, N-dipropylamino)-5,6,7,8-tetrahydro-naphtho(2,3b)dihydro-2,3-furane]. 1 These two authors made equivalent contributions to this work. 0026-895X/99/030564-11$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:564–574 (1999). 564 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from Caron, 1997; Watts and Neve, 1997). In vitro studies of agonist efficacy have employed other measures of receptor activation, including medium acification (Cox et al., 1995), and stimulation of mitogenesis (Pilon et al., 1994; Svensson et al., 1994; Sautel et al., 1995). However, these approaches measure responses “downstream” of the receptor in the intracellular activation cascade and the relevance of an increase in mitogenesis for postmitotic central nervous system neurones is unclear. A more promising approach may be to measure receptor-mediated G protein activation by stimulation of guanosine-59-O-(3-[S]thio)-triphosphate ([S]GTPgS) binding: this corresponds to the first step of the intracellular activation cascade and directly reflects ligand binding events at the receptor itself (Pregenzer et al., 1997; Malmberg et al., 1998). Thus, the present study adopted this strategy to address several questions concerning, principally, the functional properties of human (h) D3 receptors. In addition, in some tests results at hD3 receptors were compared with those at hD2 receptors. First, differences in the second-messenger actions of D3 and D2 receptors may be related to differing capacities for stimulation of G proteins. We addressed this issue by investigating the ability of hD3 receptors to mediate dopamine-stimulated [S]GTPgS binding. Second, the relationship between binding affinity and functional potency of dopaminergic agonists and antagonists was investigated using the most potent and selective D3 receptor ligands reported to date: the agonists (1)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin) and PD 128,907 [(1)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl2H,5H[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] (Pugsley et al., 1995) and the antagonists, S 14297 ((1)-[7-(N, N-dipropylamino)-5,6,7,8-tetrahydro-naphtho(2,3b)dihydro-2,3-furane]) and GR 218,231 (2(R,S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4-tetrahydronaphtalene) (Millan et al., 1995b; Murray et al., 1996). The hD3/hD2 selectivities based on Ki ratios were compared with those based on EC50 and KB ratios (Burris et al., 1995; Levant, 1997). Third, the signal transduction differences between D3 and D2 receptors, such as the differential coupling to adenylyl cyclase isoforms, could be due to receptor interactions with different G protein populations. Indeed, at least 16 distinct G protein a subunits have been identified, divided into four families: Gi, GS, Gq/11, and G12/13 (Simon et al., 1991). Although a previous study suggested differences in coupling profiles of D2 and D3 receptors for modulation of outward K currents (Liu et al., 1996), no information is available from a functional test more proximal to the receptor and the G protein subtypes involved in D3 coupling are unclear (cf. Tang et al., 1994). The present study, therefore, examined G protein coupling specificity directly at the G protein activation level by challenging the receptor-mediated stimulation of [S]GTPgS binding with specific antisera raised against different Ga subunits. In fact, antibodies raised against the COOH terminal part of Ga subunits have proved useful to determine the G protein specificity of several other 7-transmembrane domain receptors (Harris-Warrick et al., 1988; McFadzean et al., 1989; Lledo et al., 1992; Izenwasser and Côté, 1995). Materials and Methods Membrane Preparations of Chinese Hamster Ovary (CHO)hD3 and CHO-hD2 Cells. CHO cells expressing hD3 receptors were grown as described previously (Sokoloff et al., 1992). Cells were harvested from adherent culture and homogenized using a Kinematica Polytron (Kinematica GmBH, Littau, Switzerland) in a buffer containing 50 mM Tris (pH 7.4), 5 mM MgCl2. The suspension was then centrifuged at 20,000g for 15 min at 4°C and the pellet was resuspended in the appropriate binding buffer (see below) and stored Fig. 1. Saturation binding of [I]iodosulpride and [S]GTPgS to CHO-hD3 and CHO-hD2 cell membranes. A, representative saturation binding isotherms of [I]iodosulpride to CHO-hD2 and CHO-hD3 membranes. B, representative saturation binding isotherms of [ S]GTPgS to CHO-hD2 and CHO-hD3 membranes. Basal and dopamine (10 mM)-stimulated [ S]GTPgS binding were determined in the presence of increasing concentrations of GTPgS. These data were transformed as described in Materials and Methods to generate a saturation binding isotherm for net agonist-dependent [S]GTPgS binding. Points shown are means of duplicate determinations from representative experiments repeated on at least four occasions. Bmax and KD/apparent KD (Kapp) data from these experiments are shown in Table 1. TABLE 1 Densities of recombinant receptors and agonist-activated G proteins in CHO cells stably expressing hD2 and hD3 receptors Receptor expression levels (Bmax) of hD3 and hD2 receptors stably expressed in CHO cell membranes were determined by saturation binding experiments with [I]iodosulpride. Treatment of CHO-hD3 membranes with EEDQ (33 mM) significantly reduced hD3 receptor expression (p , 0.05, 2-tailed t test). Number of dopamineactivated G proteins was determined by [S]GTPgS isotopic dilution saturation binding, as described in Materials and Methods. Apparent KD for [ S]GTPgS saturation binding is denoted KAPP. EEDQ (33 mM) treatment of CHO-hD3 membranes did not significantly alter Bmax or KAPP for [ S]GTPgS saturation. Cell Line CHO-hD3 CHO-hD3 (EEDQ) CHO-hD2 Receptor Saturation Bmax (pmol/mg) 15.43 6 1.33 7.36 6 1.17 1.39 6 0.19 KD (nM) 1.18 6 0.19 1.31 6 0.03 0.48 6 0.05 G Protein Saturation Bmax (pmol/mg) 3.38 6 0.51 2.58 6 0.53 0.92 6 0.05 KAPP (nM) 6.72 6 1.29 6.18 6 1.08 1.68 6 0.30 R/G Bmax Ratio 4.6 2.9 1.5 Data are means 6 S.E.M. of at least three independent determinations. G Protein Coupling of Dopamine D3 Receptors 565 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from at 280°C. CHO-hD2(short) cell membranes were purchased from Receptor Biology (Baltimore, MD). The “short” hD2 isoform, which lacks a 29-amino acid insert in the putative third intracellular loop, is processed faster to mature receptors at the cell surface than the “long” form and may couple more efficiently to certain G protein subtypes (Fishburn et al., 1995; Boundy et al., 1996). [I]Iodosulpride Binding to hD3 and hD2 Receptors. Saturation binding at hD2 and hD3 receptors was carried out with 12 concentrations of [I]iodosulpride (1000 Ci/mmol; Amersham, Les Ulis, France). For competition binding experiments, membranes (10 to 20 mg protein) of CHO-hD2 or CHO-hD3 cells were incubated with [I]iodosulpride (0.1 nM for hD2 and 0.2 nM for hD3) at 30°C for 30 min in a buffer containing 50 mM Tris (pH 7.4), 120 mM NaCl, 5 mM KCl, 1 mM EDTA, and 5 mM MgCl2. Nonspecific binding was defined with raclopride (10 mM). Isotherms were analyzed by nonlinear regression, using the computer program PRISM (Graphpad Software Inc., San Diego, CA) to yield IC50 values. Inhibition constants (Ki values) were derived from IC50 values according to the Cheng-Prusoff equation. The goodness of fit was tested by runs test. For compounds that yielded P , .05 in the runs test and/or shallow inhibition isotherms (nH values markedly inferior to unity), 14-point competition binding experiments were carried out and oneand two-site fits were compared by F test. Measurement of Agonist Efficacy and Antagonist Potency at hD3 and hD2 Receptors. Receptor-linked G protein activation by dopamine at hD2 and hD3 receptors was determined by measuring the stimulation of [S]GTPgS (1332 Ci/mmol; NEN, Les Ulis, France) binding induced by dopamine. CHO-hD2 membranes (30–40 mg protein) were incubated (60 min, 22°C) with agonists and/or antagonists in a buffer containing 20 mM HEPES (pH 7.4), 3 mM GDP, 10 mM MgCl2, 150 mM NaCl, and 0.1 nM [ S]GTPgS. CHOhD3 membranes (30–50 mg protein) were incubated (40 min, 22°C) with agonists and/or antagonists in a buffer containing 20 mM HEPES (pH 7.4), 3 mM GDP, 3 mM MgCl2, 100 mM NaCl, and 1.0 nM [S]GTPgS. Nonspecific binding was defined with GTPgS (10 mM). Agonist efficacy is expressed relative to that of dopamine (100%), which was tested at a maximally effective concentration (10 mM) in each experiment. For all tests, membranes were preincubated with agonist and/or antagonist for 15 min before the addition of [S]GTPgS. KB values for inhibition of dopamine (1 and 3 mM for hD3 and hD2 respectively)-stimulated [ S]GTPgS binding were calculated according to Lazareno and Birdsall (1993): KB 5 IC50/{[(21(agonist/EC50) ) 21 ] 21}; where IC50 is the inhibitory concentration50 of the antagonist, agonist is the dopamine concentration, EC50 is the effective concentration50 of dopamine alone, and nH is the Hill coefficient of the dopamine stimulation isotherm. For dopamine concentration-response curves determined in the presence of fixed concentrations of the antagonist, GR 218,231, pA2 values were derived by Schild analysis. In isotopic dilution experiments, the basal and dopamine (10 mM)-stimulated binding of radiolabeled [S]GTPgS was inhibited with unlabeled GTPgS. Saturation binding curves were derived to estimate the number of G proteins activated by dopamine, as described previously (Newman-Tancredi